In phage display, a foreign peptide, domain, or protein, is fused to a structural protein and exposed on the outer surface of phage capsid (Smith, 1985). The coat proteins of the filamentous phages (M13, fd, and fl), the minor coat protein pIII (4-5 copies), and the major coat protein pVIII (2700 copies), have been extensively used to generate combinatorial libraries of six to eight amino acid long peptides (Smith and Petrenko, 1997; Manoutcharian et al., 2001). Other display systems using icosahedral phages lambda and T7 have also been developed (Maruyama et al., 1994; Danner and Belasco, 2001). These systems can display larger peptides and domains, and even full-length proteins derived from targeted clones or c-DNA libraries (Hoess, 2002). The outer capsid protein gpD (420 copies) (Stemberg and Hoess, 1995) and the tail protein gpV of phage lambda (Maruyama et al., 1994), and the major capsid protein gp10 of phage T3/T7, have been used to display foreign sequences. Rare peptides having a particular biological function can be “fished out” of these libraries by “biopanning” and then amplified (Scott and Smith, 1990; Smith and Petrenko, 1997). The connectivity between phenotype and genotype, i.e., the physical link between the peptide that is displayed on the outside of phage and the DNA that encodes it inside the same phage, allows rapid delineation of the biologically interesting peptide sequence.
Despite the availability of these display systems, significant limitations exist in the application of these systems. For example, with the filamentous phage, display of certain peptides is restricted, or not possible, since the fused peptide has to be secreted through the E. coli membranes as part of the phage assembly apparatus. Since both pIII and pVIII are essential for phage assembly, it is difficult to display large domains or full-length proteins without interfering with their essential biological functions. In situations where large peptide sequences are displayed, their copy number per phage capsid is greatly reduced and unpredictable. Similar problems on the size and copy number are encountered with the phage lambda and T3 display systems. It is often necessary to incorporate wild type protein molecules along with the recombinants to generate viable phage using either a helper phage or a partial genetic suppression of amber mutant (Hoess, 2002; Manoutcharian et al., 2001; Maruyama et al., 1994).
Another serious limitation of existing phage display systems is that they are in vivo-based in that the recombinant molecules are assembled onto the capsid as part of the phage infective cycle. In these systems, many variables in the cellular environment affect the assembly process resulting in great variability in the quality of phage particles generated. Very little control can be exerted on the assembly process and the copy number among different preparations can vary by orders of magnitude making these systems highly unpredictable.
Size and copy number of the displayed antigen are particularly critical variables for vaccine development; thus, the efforts to use phage display for creating a practical vaccine have been quite limited. An ideal phage vaccine would be capable of displaying full-length antigens or desired epitopes of an antigen at a high density without significant restrictions on size. It would also allow manipulation of the display platform in a defined way to generate particles of reproducible quality. What is needed is a first phage system that allows efficient and controlled display of full-length antigens, or epitopes of target antigens using phage T4 particles. Also desirable are phage systems that may be customized to obtain specific immune responses, for example phage systems that enable the generation of an immune response to more than one antigen or foreign particle.
The bacteriophage T4 has been explored for the development of multicomponent vaccines. The capsid of phage T4 is a prolate (elongated) icosahedron (Eiserling, 1983; Black et al., 1994) with a diameter of about 86 nm and a length of about 119.5 nm (Fokine et al., 2004; FIG. 1). It is constituted by 930 copies of a single major capsid protein, gp23* (46 kDa; blue knobs in FIG. 1). The capsid also consists of two minor capsid proteins located at the vertices. Eleven of the 12 vertices are constituted by about 55 copies (one pentamer at each vertex) of the minor capsid protein gp24* (42 kDa; magenta knobs in FIG. 1). The twelfth vertex is constituted by about twelve identical copies (dodecahedron) of the minor capsid protein gp20 (61 kDa; not shown in FIG. 1). This vertex is also referred to as the portal vertex since it serves both as an entry point and as an exit point for T4 DNA.
Structural studies have established that two additional proteins, namely Hoc (Highly antigenic outer capsid protein, 40 kDa) and Soc (Small outer capsid protein, 9 kDa), (FIG. 1) are added onto the capsid after completion of capsid assembly (Steven et al., 1976; Yanagida, 1977; Ishii and Yanagida, 1975 and 1977; Ishii et al., 1978, Iwasaki et al., 2000). According to the most recent structural data reported by Fokine et al. (2004), Hoc is present up to 155 copies per capsid particle, whereas Soc is present up to 810 copies per capsid particle. Most importantly, these proteins are nonessential. Mutations in either of the genes, or in both the genes, do not affect phage production, phage viability, phage infectivity, or phage stability under normal experimental conditions. However, Hoc and Soc provide additional stability to the capsid under extreme environmental conditions (eg., pH>10.6, osmotic shock).
When others first reported Hoc and Soc, it was thought that these proteins represented a new and interesting class of outer capsid proteins that form an outer “cage/armor” to protect the virus in its extracellular phase of the life cycle. Yet, since their discovery, no other phage/virus system has been shown to possess such non-essential, high copy number, highly antigenic, relatively easily manipulable, outer capsid genes.
One useful feature of Hoc and Soc proteins is that one can fuse foreign proteins or protein fragments to the N- and C-termini of Hoc and Soc without affecting T4 phage function. In fact, display of Hoc and Soc fusion proteins does not affect phage viability or infectivity (Jiang et al., 1997; Ren et al., 1996; Ren and Black, 1998). Large polypeptide chains and full-length proteins have been fused to Hoc and Soc and successfully displayed on the T4 capsid surface. These include the Por-A loop-4 peptide (4 kDa), HIV-gp120 V3 loop (5 kDa), soluble CD4-receptor (20 kDa), anti-egg white lysozyme domain (32 kDa), and poliovirus VP1 (35 kDa), (Jiang et al., 1997; Ren et al., 1996; Ren and Black, 1998). Furthermore, the foreign proteins were stably displayed on the capsid, and can be stored for several weeks at 4° C., or in the presence of high salt concentration (Jiang et al., 1997; Ren et al., 1996). The T4 recombinant nanoparticles elicited high titer antibodies in mice against the displayed antigens.
Previous strategies have utilized an unpredictable in vivo loading of foreign proteins onto the phage capsid. This has been the prevailing paradigm in the phage display field using phages M13, lambda, T7 and T4. In one in vivo strategy, the proteins are first expressed in E. coli and then loaded onto T4 following infection with hoc−soc− virus (Jiang et al., 1997). In a second in vivo strategy, the fusion construct is transferred into the T4 phage genome by recombinational exchange and the fusion protein is expressed and loaded onto phage T4 during the course of T4 infection; in this strategy, the recombinant gene and gene product become a part of phage T4 life cycle (Jiang et al., 1997; Ren et al., 1996). A major drawback of the in vivo loading systems is the variability in the copy number of the displayed antigen. This is largely due to variation of antigen assembly in vivo upon which little control can be exerted. For example, the expression level of recombinant antigen in the infected cell varies greatly depending upon nutritional and environmental conditions. Also, the assembly process is susceptible to nonspecific intracellular proteolysis. Additionally, interactions among numerous components of the intracellular milieu make it a poorly defined process for producing homogeneous particles with consistent quality.
Various Hoc and Soc-based assembly platforms have been conceptualized. For example, in U.S. Pat. No. 6,500,611 issued to Mattson, the inventor describes a general concept for linking a reporter group to a viral capsid wherein the reporter group recognizes an analyte via a linker molecule. Mattson, however, fails to enable specific methods for loading foreign proteins onto a T4 phage capsid. Also, Mattson fails to demonstrate or suggest that large full-length capsid proteins can be loaded at a high density on the capsid surface. Moreover, Mattson fails to teach or suggest T4 nanoparticle vaccine compositions or that any such compositions may be used as a multicomponent platform for eliciting an immunogenic response.
In studies by Ren et al., Protein Science, September; 5(9), 1833-43 (1996), the authors discuss the binding of Soc fusion proteins to capsid-based polymers called polyheads. This polyhead model is particularly unsuited for development of defined assembly platforms and vaccine compositions. Foremost, polyheads are not defined particles. Rather, these polymers result from the uncontrolled growth of phage T4 major capsid protein gp23 and exist as a heterogeneous mixture of particles after their preparation. For example, to even posses Hoc and Soc binding sites, one must cleave polyheads polymers in vitro in the presence of a crude extract containing the phage T4 prehead protease in order to open up the binding sites for Hoc and Soc. The latter also requires “polyhead expansion”, a dramatic conformational change that reorganizes the capsid protein polymer and creates the Hoc and Soc binding sites. The resulting cleaved, expanded, polyheads will have ill-defined number of Hoc and Soc binding sites on a structurally heterogeneous mixture of polyheads, whose length can vary anywhere from a few nanometers to micrometers. Unlike T4 phage particles, these polyheads comprise flat, two-dimensional structures; they contain sheets, closed sheets (tubes), and broken pieces of gp23 polymers, etc. of varying size and dimensions. Given this variability of the polyhead model, the number of available binding sites on the particles cannot be determined accurately with undue experimentation. Thus, controlling the copy number of a foreign antigen on the polyheads would be extremely difficult if not impossible. Also, because of their shape, polyheads are not competent to package DNA and can thus not be used a prime-boost strategies known in the art.
What is needed are effective compositions and methods for customizing bacteriophages. Customized bacteriophages may be used to create vaccine systems comprising customized phage particles. Such systems should enable the design of specific phage particles capable of eliciting an immune response to one or more antigens or foreign particles. Preferably, such a system should be easy to manufacture and administer.
What is also needed are compositions and methods to target the exposure or delivery of specific antigens or particles to target cells.
There is also a general need for compositions and improved methods for producing antibodies. These compositions and methods should be easily and economically produced in a manner suitable for therapeutic and diagnostic formulation.